Matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) has become an increasingly common tool for protein analysis in biological research since its development in 1988 (Karas, et al 1988, Tanaka et al RCMS, Fenseleau). The simple sample preparation, short analysis time and sensitivity have made this a powerful technique for protein identification (Fenseleau, Anal. Chem. 1997). Furthermore, the ability to generate intact molecular ions for whole proteins directly from complex mixtures makes this a particularly attractive technique for biological samples. (Redeker et al Anal Chem 1998). Electrospray ionization has proven to be another powerful and widespread ionization technique for mass spectrometric analysis of proteins and peptides that provides a means to directly couple liquid separations and mass analysis (Washburn, M. P., D. Wolters, and J. R. Yates III Nat. Biotech. 2001xe2x80x94Veenstra, T. D., S. Martinovic, G. A. Anderson, L. Pasa-Tolic, and R. D. Smith JASMS 2000). However the necessity of a liquid phase for the samples prior to ionization is in contrast to MALDI-MS where the sample is generally allowed to dry on a surface in combination with matrix molecules. It is the ability to generate ions from a solid phase sample that has led to a unique application of this technique whereby the location of the analyte within a heterogenous sample can be determined along with its mass.
MALDI-MS has been used to obtain mass spectra for proteins and peptides from precise X-Y locations from within a complex biological sample such as single cells (Garden et al JMS 1996xe2x80x94Chaurand, Stoeckli, and Caprioli Anal Chem 1999.). An extension of this approach was later described where multiple mass spectra were obtained by rastoring across the sample in a grid like pattern across the sample to form the pixels of an image.xe2x80x94(Stoeckli, M., T. B. Farmer, and R. M. Caprioli JASMS 1999xe2x80x94Caprioli, Farmer, Gile Anal Chem 1997) Using this data, the mass spectra could then be reassembled to create an image detailing the two dimensional position for a particular m/z value and therefore the corresponding protein. This has been demonstrated with several types of tissues and most dramatically with tissue sections from a rat brain (Todd et al, 2001, Stockle et al 2001-Stoeckli, Caprioli Nat. Med. 2001).
Other types of desorption/ionization mass spectrometry have been used to generate an xe2x80x9cion imagexe2x80x9d, but have generally used relatively harsh ionization methods, such as secondary ion mass spectrometry and laser ablation, and were limited to examining low molecular weight species (Belu, A. M. et al Anal Chem. 2001, Todd et al, 2001,xe2x80x94Stockle et al 2001-Kossokovski et al 1998). Two of these reports achieved a very tightly focused laser beam with near field microscopy fibers (Stockle et al 2001-Kossokovski et al 1998). However, one limitation of the near field microscopy approach is the fluence achievable at the fiber optic tip. Fiber optic damage thresholds are too easily exceeded when the tip diameter is reduced to 150-200 nm. Precision control of laser intensity and beam profile is required to inhibit fiber optic tip heating and self-ablation. In addition, because of the necessity for the fiber optic tip to be in the proximity of the desorption surface; contamination of the tip surface is a constant concern. Coupled with the effect of laser heating, tip lifetime is compromised.
Several challenges to creating an image from mass spectral data have been identified in prior work. Among them, are the need to evenly distribute the matrix over the sample to generate a homogeneous surface, removal of contaminant peaks that may suppress the signal of other analytes, and visualization of the tremendous amount of data generated by this technique. These issues have been described in a recent review (Todd, P. J and R. M. Caprioli. JMS 2001). One limitation that exists is the resolution that can be achieved when creating the xe2x80x9cprotein imagexe2x80x9d. The picture resolution is limited by the pixel size achievable, which is in turn limited by the size of the laser spot used to perform the ionization. Typically the laser spot size used to obtain MALDI-MS spectra is on the order of 25 xcexcm in diameter (Stockle, R., R. Zenobi Anal Chem) with limitations at the 1 xcexcm level mentioned (Todd et al 2001). However, the laser spot size reliably used for MALDI imaging has remained at 5 to 100 xcexcm. (ibid.). While this provides ample resolution to distinguish structures within tissue sections or single neurons from A. californica (cells on the order of 92 xcexcm), smaller structures/cells cannot be resolved from one another using this pixel size (Rubakhin, S. S. et al J. Neurophys 1999). In particular, microbes are often on the order of 1-2 xcexcm in length and great resolution is required (Auerbach, I. D. et al J. Bact. 2000). Reductions in laser spot size are needed in order to generate MALDI-MS images of cells and extracellular structures on the microbial scale.
An improvement in laser focus can also lead to additional benefits by improving the ability of MALDI-MS to ionize extremely small protein samples in the analysis of dense protein arrays. Currently, sample plates holding up to 384 sample wells (each xcx9c2 mm in diameter), are used for high throughput protein analysis using MALDI-MS. Manufacturers have introduced sample plates such as the xe2x80x9cAnchor chip(trademark)xe2x80x9d with affinity or adsorptive surfaces to concentrate the sample on a small area (Bruker Daltonics Inc., Product information literature, 2001). These aid in concentrating the sample as well as potentially creating more tightly packed arrays of samples with smaller spots from 200 to 800 xcexcm in diameter. Meanwhile there have been other notable developments in deposition of small sample spots for analytical arrays that may have application MALDI-MS protein analysis. Methods creating small protein spots by spray deposition have been described with spots ranging from 100 to 500 microns (Onnerfjord, P et al. Anal Chem 1998xe2x80x94Moerman, R., et al. Anal. Chem.2001). Furthermore, microstructured devices have been fabricated as microreactors with features on the 1 to 5 xcexcm scale with reactor wells of 15 xcexcm being produced (Grzybowski, B. A., R. Haag, N. Bowden, and G. M. Whitesides, Anal. Chem 1998). While these spots are still above the typical laser spot size used for MALDI-MS, a recent report of protein samples being deposited in 15 nm diameter spots has appeared (Perkel, C. The Scientist 2002,16[5], p.34,). Clearly, as technologies for depositing arrays of samples improve, methods for producing smaller laser spots for both ionization and imaging in association with MALDI-MS are needed.
The development and application of tightly focused MALDI in the present invention allows for generation of higher resolution images detailing intact protein location and the creation of xe2x80x9cprotein imagesxe2x80x9d of a small sample area that can be compared to optical images to reveal their location within a 2-D sample.
One object of the present invention provides a system and process for focusing light to a spot size for matrix assisted laser desorption/ionization (MALDI). A coherent light source, such as a laser or infrared light source, may be directed through at least one confocal microscopic objective to create a desorption/ionization source at the surface of a MALDI sample plate adapted to receive a sample substrate within the focal working distance of the microscopic objective. The light is transported by at least one fiber optic cable to at least one confocal microscopic objective. At least one collimating fiber optic coupler is employed to collimate the light to an aperature of at least one fiber optic cable. A insulating microscopic objective holder holds the microscopic objective and insulates it from the electrical fields of the MALDI. At least one adapter secures the insulating microscopic objective holder. At least one X, Y, positioner moves the microscopic objective in the X, Y co-ordinates. At least one Z positioner moves the microscopic objective in the Z co-ordinate. Finally, a mass analyzer is used to analyze ions desorbed from the sample substrate.
A preferred embodiment of the present invention provides a system and process for focusing light to a submicron spot size for matrix assisted laser desorption/ionization (MALDI). A coherent light source, such as a laser, is used to generate ultra-violet light. At least one confocal microscopic objective is used to create a desorption/ionization source of sub-micron spatial resolution at the surface of a MALDI sample plate. The ultra-violet light generated by the laser is transported by at least one fiber optic cable to at least one confocal microscopic objective. A sample substrate is placed on a sample plate to hold it within the focal working distance of the microscopic objective and a mass analyzer is used to analyze the sample after it has been ionized.
As used herein, a sample substrate is a combination of an analyte and an appropriately absorbing sample matrix. As used herein, working distance includes the distance from the front lens element of the objective to the closest surface of the coverslip when the specimen is in sharp focus. In the case of objectives designed to be used without coverslips, the working distance is determined by the linear measurement of the objective from lens to the specimen surface. As used herein, a mass analyzer is any device capable of separating and detecting ions based upon their mass to charge (m/z) ratio. It includes mass spectrometer devices operated under vacuum (from 760 torr down to 10xe2x88x929 torr), such as a time-of-flight mass spectrometer, and devices operated at or near atmospheric pressure (e.g. an ion mobility spectrometer).
In another arrangement of the system, at least one confocal microscopic objective is positioned above the slide.
Optionally, the slide may be transparent and at least one confocal microscopic objective is positioned below the transparent slide.
In a further arrangement of the system, the mass analyzer includes at least one ion mobility spectrometer alone or in tandem with a mass spectrometer.
In still another arrangement of the system, the mass analyzer includes a mass spectrometer having at least one evacuated internal chamber.
In still another further arrangement of the system, the confocal microscopic objective and sample plate in either of the prior-mentioned arrangements may be positioned outside of a vacuum chamber. In this arrangement, the ions produced from the slide are transmitted into the evacuated chamber of the mass analyzer.
The present invention also features a process for focusing a light source to a micron and sub-micron spot sizes for matrix assisted laser desorption/ionization (MALDI), including the steps of (i) depositing a sample substrate containing analyte and an appropriately absorbing matrix on a sample plate; (ii) generating a coherent light source; (iii) positioning the sample plate within the focal working distance of at least one confocal microscopic objective; (iv) positioning at least one confocal microscopic objective in a geometry which does not interfere with the path of desorbed sample ions; (v) coupling at least one confocal microscopic objective to the coherent light source, such as a laser, with at least one fiber optic cable; (vi) focusing the coherent light source at least one microscopic objective to create a desorption/ionization laser source of submicron and micron spatial resolution at the sample substrate; (vii) ionizing the sample substrate; (viii)separating and detecting ions from the ionized sample substrate in one or more stages using an appropriate mass separation and analysis method.
In another arrangement of the process, at least one confocal microscopic objective is positioned above the sample plate.
Optionally, the sample plate may be transparent and at least one confocal microscopic objective is positioned below the transparent sample plate.
In a further arrangement of the process, the mass analyzer includes at least one ion mobility spectrometer alone or in tandem with a mass spectrometer.
In still another arrangement of the process, the mass analyzer includes at least one evacuated internal chamber, such as a mass spectrometer.
Moreover, the present invention features a second process for creating a correlated optical image of the ion desorption region of a sample substrate. The process may include (i) depositing a solution containing an analyte and an appropriately absorbing matrix on a MALDI sample plate; (ii) generating a coherent light source; (iii) positioning the sample plate within the focal working distance of at least one confocal microscopic objective; (iv) coupling at least one confocal microscopic objective to the coherent light source, such as a laser, with at least one fiber optic cable; (v) positioning at least one confocal microscopic objective in a geometry which does not interfere with the path of desorbed sample ions; (vi) focusing the coherent light through said at least one microscopic objective to create a desorption/ionization ultra-violet light source of submicron spatial resolution directed at said sample substrate; (vii) ionizing the sample substrate; (viii) illuminating the sample substrate; (ix) transferring an optical image of the ionized sample substrate using said at least one fiber optic cable; and (x) separating and detecting desorbed ions from said ionized sample substrate in one or more stages using an appropriate mass separation and analysis method.
Furthermore, the present invention also includes a system for creating a correlated optical image of the ion desorption region of a sample substrate. The system may include a) a coherent light source, such as a laser, b) at least one confocal microscopic objective to create a desorption/ionization source of sub-micron spatial resolution at the surface of a MALDI sample plate, c) a sample substrate and a sample plate to hold the sample substrate, d) a device for capturing an optical image of the sample substrate, such as a charged coupled device (CCD) camera and f) an image display unit operatively connected to the optical imaging device through a fiber optic cable or other means.